Purification and Characterization of Proteasomes from Saccharomyces cerevisiae

Michael Glickman1, Olivier Coux2

1 The Technion, Haifa, 2 CRBM‐CNRS, Montpellier
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.5
DOI:  10.1002/0471140864.ps2105s24
Online Posting Date:  August, 2001
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Abstract

The proteasome plays a central role in eukaryotic cells since it is responsible for the degradation of specific proteins involved in a large range of cellular processes. Analysis of proteasome mechanisms of action, or in vitro reconstitution, or dissection of the complex biological pathways in which it partakes, requires a reliable source of pure active proteasome. Although the biologically relevant form of the proteasome is usually considered to be the 26S proteasome, this unit describes different methods for purification and study of both 26S and 20S proteasomes from Saccharomyces cerevisiae cells.

     
 
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Table of Contents

  • Basic Protocol 1: Purification of Yeast 26S Proteasome by Anion‐Exchange Chromatography and Gel Filtration
  • Alternate Protocol 1: Purification of Yeast 26S Proteasome by Affinity Chromatography
  • Basic Protocol 2: Purification of Yeast 20S Proteasome by Conventional Chromatography
  • Support Protocol 1: Peptidase Activity Assay for 26S and 20S Proteasome
  • Support Protocol 2: Preparation of Poly‐Ubiquitinated Lysozyme
  • Support Protocol 3: Preparation of Radiolabeled Casein
  • Support Protocol 4: Degradation of Protein Substrates by the 26S Proteasome
  • Support Protocol 5: Native Gel Electrophoresis/in‐Gel Peptidase Assay of the Proteasome
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Yeast 26S Proteasome by Anion‐Exchange Chromatography and Gel Filtration

  Materials
  • Saccharomyces cerevisiae (e.g., pep4) cells
  • YPD ( appendix 4L) or other appropriate medium
  • recipe26S lysis buffer (see recipe)
  • 1 M Tris base
  • DEAE Affi‐Gel Blue resin (Bio‐Rad)
  • recipe26S chromatography buffer (see recipe) with and without 50, 100, 150, and 500 mM NaCl
  • High‐resolution “Q” anion‐exchange resin (e.g., Pharmacia Mono‐Q or Resource Q; Bio‐Rad Bio‐scale Q); also see unit 8.2
  • Gel‐filtration resin with high‐molecular‐weight resolution (e.g., Sephacryl S‐400HR or Superose 6HR; Amersham/Pharmacia); also see unit 8.3
  • French press or equivalent
  • Cheesecloth
  • Appropriately sized chromatography columns (see )
  • FPLC‐type chromatography system
  • Additional reagents and equipment for testing peptidase activity (see protocol 4) and performing ion‐exchange (unit 8.2) and gel‐filtration chromatography (unit 8.3)

Alternate Protocol 1: Purification of Yeast 26S Proteasome by Affinity Chromatography

  Materials
  • Saccharomyces cerevisiae (e.g., pep4) cells
  • YPD ( appendix 4L) or other appropriate medium
  • recipe20S lysis buffer (see recipe)
  • 1 M Tris base
  • High‐capacity anion‐exchange resin (e.g., Q‐Sepharose or Q‐Fast Flow; Amersham); also see unit 8.2
  • 25 mM Tris⋅Cl, pH 7.4 ( appendix 2E), with 100 mM, 200 mM, and 1 M NaCl
  • 1 M MgCl 2 ( appendix 2E)
  • Hydroxyapatite column (Bio‐Rad); also see unit 8.6
  • 40 and 400 mM potassium phosphate buffer pH 7.2 ( appendix 2E)
  • High molecular weight–resolving gel (e.g., Sephacryl S300HR or Superose 6HR; Amersham); also see unit 8.3
  • French press or equivalent
  • Cheesecloth
  • Appropriately sized chromatography columns (see )
  • Additional reagents and equipment for ion‐exchange (unit 8.2), gel‐filtration (unit 8.3), and hydroxylapatite chromatography (unit 8.6), and testing peptidase activity (see protocol 4) or performing the native gel procedure (see protocol 8)

Basic Protocol 2: Purification of Yeast 20S Proteasome by Conventional Chromatography

  Materials
  • Protein fractions to be analyzed (see Basic Protocols protocol 11 and protocol 32 and protocol 2)
  • 10 mM N‐suc‐LLVY‐AMC (Bachem or Sigma) in DMSO
  • recipeActivity buffer (see recipe)
  • 1% (w/v) SDS
  • 1‐ml quartz or appropriate type of plastic cuvette
  • Fluorometer with 380‐nm excitation and 440‐nm emission wavelength

Support Protocol 1: Peptidase Activity Assay for 26S and 20S Proteasome

  Materials
  • 15 mg/ml β‐casein in H 2O
  • 200 µl 1 M potassium phosphate buffer, pH 8 ( appendix 2E)
  • 250 µCi [14C]formaldehyde in ampule (ICN)
  • 200 µl 0.2 M sodium cyanoborohydride (NaCNBH 3)
  • Small desalting column (e.g., PD‐10 column; Pharmacia)
  • Additional equipment and reagents for dialysis ( appendix 3B)

Support Protocol 2: Preparation of Poly‐Ubiquitinated Lysozyme

  • Enzyme (see Basic Protocols protocol 11 and protocol 32 and protocol 2)
  • Radioactive substrate (see Support Protocols protocol 52 and protocol 63)
  • recipeActivity buffer (see recipe)
  • Carrier protein (e.g., BSA)
  • 10% or 20% TCA
  • Appropriate instrument for measuring radioactivity

Support Protocol 3: Preparation of Radiolabeled Casein

  Materials
  • recipe1.5‐mm‐thick 4% polyacrylamide gel (see recipe)
  • recipe1× native gel electrophoresis buffer, pH 8.1 to 8.4 (see recipe)
  • 5× native gel loading buffer: 50% glycerol to which a tiny speck of xylene cyanol is added
  • Proteasome sample (see Basic Protocols protocol 11 and protocol 33 and protocol 2)
  • recipeNative gel assay buffer (see recipe)
  • Electrophoresis apparatus
  • UV lamp
  • Camera and appropriate filters
  • Additional equipment and reagents for casting and running native acrylamide gels (unit 10.3), and staining gels with Coomassie blue (unit 10.5)
NOTE: All equipment and reagents must be prechilled to 4°C.
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Figures

Videos

Literature Cited

Literature Cited
   Beers, E.P. and Callis, J. 1993. Utility of polyhistidine‐tagged ubiquitin in the purification of ubiquitin‐protein conjugates and as an affinity ligand for the purification of ubiquitin‐specific hydrolases. J. Biol. Chem. 268:21645‐21649.
   Braun, B.C., Glickman, M., Kraft, R., Dahlmann, B., Kloetzel, P.M., Finley, D., and Schmidt, M. 1999. The base of the proteasome regulatory particle exhibits chaperone‐like activity. Nat. Cell Biol. 1:221‐226.
   Chu‐Ping, M., Vu, J.H., Proske, R.J., Slaughter, C.A., and Demartino, G.N. 1994. Identification, purification, and characterization of a high molecular weight, ATP‐dependent activator (PA700) of the 20‐S proteasome. J. Biol. Chem. 269:3539‐3547.
   Ciechanover, A., Elias, S., Heller, H., and Hershko, A. 1982. “Covalent affinity” purification of ubiquitin‐activating enzyme. J. Biol. Chem. 257:2537‐2542.
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   Rock, K.L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A.L. 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class 1 molecules. Cell 78:761‐771.
   Rubin, D.M. and Finley, D. 1995. Proteolysis. The proteasome: A protein‐degrading organelle. Curr. Biol. 5:854‐858.
   Rubin, D.M., Coux, O., Wefes, I., Hengartner, C., Young, R.A., Goldberg, A.L., and Finley, D. 1996. Identification of the Gal4 suppressor Sug1 as a subunit of the yeast 26S proteasome. Nature 379:655‐657.
   Seemuller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. 1995. Proteasome from thermoplasma acidophilum—a threonine protease. Science 268:579‐582.
   Tamura, T., Tanaka, K., Tanahashi, N., and Ichihara, A. 1991. Improved method for preparation of ubiquitin‐ligated lysozyme as substrate of ATP‐dependent proteolysis. FEBS Lett. 292:154‐158.
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